Summary The goal of this proposal is to understand whether signaling from CD27, a member of the tumor necrosis factor receptor superfamily, regulates hematopoietic stem cell formation in the embryo. Understanding how HSCs form, and in particular the signaling pathways that are involved in embryonic HSC formation will be essential for producing and expanding HSCs from other cell sources ex vivo, which is a major goal in regenerative medicine. All adult HSCs are derived from hemogenic endothelial cells in the embryo. Hemogenic endothelium is located in several anatomic sites, but only hemogenic endothelium in the major arteries produces HSCs and immature precursors of HSCs called pre-HSCs. We and others discovered that multiple innate immune and inflammatory signaling pathways regulate HSC formation in the major arteries. We have also identified a cell surface receptor, CD27, expressed on pre-HSCs and HSCs. In the adult, CD27 activates inflammatory signaling through the NF-?B and JNK pathways. We will test the hypothesis that CD27 signaling regulates NF- ?B signaling and the number of pre-HSCs and HSCs in the embryo. We will also determine the identity of the cells expressing CD70, the ligand for CD27.